Integration of Murine Retroviral Vectors

描述(由申请人提供)：基因治疗方案有可能由于逆转录病毒插入激活而激活基因。然而，如果没有对逆转录病毒整合的基本了解，就不可能成功地实现重定目标或限制逆转录病毒整合部位的能力。这项建议研究了小鼠白血病病毒(MuLV)的整合，这是基因治疗方案中最常见的逆转录病毒载体。整合是病毒生命周期中的关键步骤，它导致病毒基因组在宿主DNA中稳定插入。整合的前病毒没有切除的机制，整合可能导致细胞基因的破坏和/或激活。该提案利用生化和遗传方法来定义协调蛋白质-DNA整合复合体组装的相互作用。提出了三个具体目标，以审查整合综合体的各个组成部分。第一个目的是研究病毒整合酶(IN)蛋白，它催化酯交换过程。这些研究旨在确定与病毒和靶DNA组装突触复合体所需的蛋白质结构域。第二个特定目的定义了病毒底物的结构和/或序列要求，反向末端在LTRS的末端重复。已经开发出序列特异性结合在MuLV LTR末端的小凹槽内的新型聚酰胺。我们将研究这些化合物对MuLV LTR结构的影响。第三个特定目的是修改MuLV IN蛋白，使其包括被发现直接整合到异染色质的序列，这些序列被认为是转录不活跃的。这种方法将使酵母TY5系统所确定的机制适应逆转录病毒颗粒。将研究TY5标签靶向整合逆转录病毒的潜力。到目前为止，还没有已知的带有靶标或病毒DNA的逆转录病毒整合酶的结构。这些研究检查了识别稳定的突触复合体用于结构和功能分析所需的蛋白质-DNA识别的关键方面。这种逆转录病毒整合的知识对于进一步操作以靶向和/或抑制病毒生命周期中的这一必要步骤是必要的。

DESCRIPTION (provided by applicant): Gene therapy protocols risk the potential of gene activation due to retroviral insertional activation. The ability to retarget or limit the sites of retroviral integration though cannot be successfully achieved in the absence of the basic understanding of retroviral integration. This proposal studies the integration of murine leukemia virus (MuLV), the most common retroviral vector used in gene therapy protocols. Integration is a critical step in the viral life cycle which results in the stable insertion of the viral genome throughout the host DNA. There is no mechanism of excision of an integrated provirus and integration can lead to the disruption and/or activation of cellular genes. The proposal utilizes biochemical and genetic approaches to define the interactions which orchestrate the assembly of protein-DNA integrative complex. Three specific aims are proposed which examine individual components of the integration complex. The first aim focuses on the viral Integrase (IN) protein, which catalyzes the transesterification process. These studies aim at defining the domains of the protein required to assemble a synaptic complex with the viral and target DNAs. The second specific aim defines the structural and/or sequence requirements of the viral substrate, the inverted terminal repeats at the ends of the LTRs. Novel polyamides which sequence specifically bind within the minor groove of the MuLV LTR termini have been developed. The effect of these compounds on the structure of the MuLV LTR will be examined. The third specific aim modifies the MuLV IN protein to include sequences found to direct integration to heterochromatin, considered to be transcriptionally inactive. This approach will adapt the mechanism identified for the yeast Ty5 system to the retroviral particles. The potential of the Ty5 tag to target retroviral integration will be examined. To date, there is no known structure of a retroviral integrase with either the target or viral DNA. These studies examine critical aspects of the protein-DNA recognition needed to identify a stable synaptic complex for structural and functional analysis. This knowledge of retroviral integration is necessary for further manipulation to target and/or inhibit this requisite step in the viral life cycle.